Mass Spectrometry Detection of Drugs in Single Bladder Cancer Cells from Patients Cancer is increasingly understood as a process defined and propagated at the single cell level. Research in cancer stem cell biology, chemotherapeutic resistance development, and metastatic disease all focus on the action of individual cancer cells. To approach cancer research and therapy on the single cancer cell level, new bioanalytical capabilities and technologies are needed. A major unmet bioanalytical need at the interface of personalized medicine and single cell analysis will be the capability to monitor the dosing and effectiveness of patient-administered chemotherapeutic therapies on the single cancer cell level. Currently, there are no clinical bioanalytical methods capable of determining the concentration of chemotherapeutic agents inside of a patient's individual cancer cells, and such a method would be a powerful tool in establishing ideal dosing regiments that deliver effective chemotherapeutic concentrations with minimal toxicities. Further, a single cell analysis method that could also assess the effectiveness of the patient administered chemotherapeutic on the health of the individual cancer cells (e.g. apoptosis level) would give real-time relevant information about the therapeutic efficacy. We have developed a novel device-the Single-probe-that can be coupled with mass spectrometry (MS) for bioanalysis. The Single-probe, with a sampling tip smaller than eukaryotic cells (<10 ?m), can be inserted into individual living cancer cells to sample the intracellular compounds for immediate MS analysis. In our published results, we have proven that this technique can be used to detect the presence of anti-cancer compounds and their metabolites inside of single cancer cells cultured and dosed in vitro. Our long term research goal is to fully develop the Single-probe MS technique as a bioanalytical method to improve the effectiveness of chemotherapy in patients. The objective of this application is to establish protocols to use the Single-probe MS technique for quantitative single cell MS (qSCMS) of chemotherapeutic agents in patient isolated cancer cells. This research objective will be accomplished through completing the following two Specific Aims: Aim 1: Using the Single-probe technology, we will establish an experimental protocol for the qSCMS of several anti-cancer compounds, including standard of care (SOC) drugs, inside bladder cancer cells. Aim 2: We will apply the in vitro qSCMS protocol developed in Aim 1 to quantitate the intracellular levels of chemotherapy drugs in bladder cancer cells isolated from the urine of treated Stephenson Cancer Center patients. The proposed research is novel and significant. It will be the first time to quantitatively measure anti-cancer drugs delivered into patient's cancer cells. The success of this project will allow for new understandings in single cell and tumor biology that are not currently possible.